The activation of proteins by post-translational modification represents an important cellular mechanism for regulating most aspects of biological organization and control, including growth, development, homeostasis, and cellular communication. For example, protein phosphorylation plays a critical role in the etiology of many pathological conditions and diseases, including cancer, developmental disorders, autoimmune diseases, and diabetes. In spite of the importance of protein modification, it is not yet well understood at the molecular level. The reasons for this lack of understanding are, first, that the cellular modification system is extraordinarily complex, and second, that the technology necessary to unravel its complexity has not yet been fully developed.
The complexity of protein modification, including phosphorylation, on a proteome-wide scale derives from three factors: the large number of modifying proteins, e.g. kinases, encoded in the genome, the much larger number of sites on substrate proteins that are modified by these enzymes, and the dynamic nature of protein expression during growth, development, disease states, and aging. The human genome encodes, for example, over 520 different protein kinases, making them the most abundant class of enzymes known. See Hunter, Nature 411: 355-65 (2001). Each of these kinases phosphorylates specific serine, threonine, or tyrosine residues located within distinct amino acid sequences, or motifs, contained within different protein substrates. Most kinases phosphorylate many different proteins: it is estimated that one-third of all proteins encoded by the human genome are phosphorylated, and many are phosphorylated at multiple sites by different kinases. See Graves et al., Pharmacol. Ther. 82:111-21 (1999).
Many of these phosphorylation sites regulate critical biological processes and may prove to be important diagnostic or therapeutic targets for molecular medicine. For example, of the more than 100 dominant oncogenes identified to date, 46 are protein kinases. See Hunter, supra. Oncogenic kinases such as ErbB2 and Jak3, widely expressed in breast tumors and various leukemias, respectively, transform cells to the oncogenic phenotype at least in part because of their ability to phosphorylate cellular proteins. Understanding which proteins are modified by these kinases will greatly expand our understanding of the molecular mechanisms underlying oncogenic transformation. Thus, the ability to identify modification sites, e.g. phosphorylation sites, on a wide variety of cellular proteins is crucially important to understanding the key signaling proteins and pathways implicated in disease progression, for example cancer.
The efficient identification of protein phosphorylation sites relevant to disease has been aided by the recent development of a powerful new class of antibodies, called motif-specific, context-independent antibodies, which are capable of specifically binding short, recurring signaling motifs comprising one or more modified (e.g. phosphorylated) amino acids in many different proteins in which the motif recurs. See U.S. Pat. No. 6,441,140, Comb et al. Many of these powerful new antibodies are now available commercially. See CELL SIGNALING TECHNOLOGY, INC. 2003-04 Catalogue. More recently, a powerful new method for employing such motif-specific antibodies in immunoaffinity techniques coupled with mass spectrometric analysis to rapidly identify modified peptides from complex biological mixtures has been described. See U.S. Patent Publication No. 20030044848, Rush et al.). Such techniques will enable the rapid elucidation of protein activation and phosphorylation events underlying diseases, like cancer, that are driven by disruptions in signal transduction.
Human c-Src, a non-receptor tyrosine kinase, is one such signaling molecule that is over-expressed and activated in large number of human cancers. Increased c-Src activity has been demonstrated in a variety of human cancers, including breast, colon, pancreatic, ovarian, lung, esophogeal, and neural. See, e.g., Yeatman, Nature Reviews 4: 470-480 (2004); Irby et al., Oncogene 19: 5636-642 (2000). In addition to its role in regulating cell proliferation, c-Src contributes to later-stage metastatic potential of cells via effects on adhesion, invasion, and motility. See, e.g., Yeatman supra.
Human c-Src kinase activity is regulated via phosphorylation of two critical tyrosine residues, Tyr419 and Tyr530. Autophosphorylation at Tyr419 in the SH1 kinase domain is required for full c-Src activity. Tyr530 in the c-terminal tail is involved in the down-regulation of c-Src. Phosphorylation of Tyr530 leads to a conformational change involving C-terminal binding to the SH2 domain, which results in diminished substrate access to the catalytic kinase domain and thus, reduced c-Src activity. See Yeatman, supra; Irby et al., supra. Accordingly, phosphatases that de-phosphorylate c-Src at the regulatory Tyr530 site can activate this kinase even at normal expression levels.
It is known that c-Src can be activated by a number of upstream receptor tyrosine kinases, including EGFR, PDGFR, ERBB2, and FGFR, among others, and interactions with these ligand-activated receptors can lead to synergistic c-SRC activation. Additionally, a number of downstream signaling protein targets of activated c-Src have been identified as potentially involved in mediating cellular transformation, including FAK (itself a non-receptor tyrosine kinase involved in regulating cell-cycle progression, survival, and migration), p190 RhoGAP, p120 RasGAP, and cortactin, whose association with, and/or phosphorylation by c-Src leads to cellular adhesion disassembly. See Yeatman, supra; Irby et al., supra. ERK is also a target of c-Src/FAK signaling, and its phosphorylation results in activation of MLCK, which contributes to adhesion disassembly. See Yeatman, supra. Activated c-Src is known to activate the transcription factor, STAT3. See Irby et al., supra. It is also believed that c-Src activation impacts metalloproteinase function, and hence the invasive potential of cells, via the c-JUN kinase signaling pathway. c-Src also induces VEGF activity, leading to enhanced angiogenesis. See Irby et al, supra.
However, despite the identification of some of the downstream targets of c-Src, the molecular mechanisms contributing to c-Src-mediated oncogenesis in a variety of human cancers remain incompletely understood. See Yeatman, supra. Indeed, while interest in c-Src as a therapeutic target has recently increased—Wyeth (SKI-606), Sugen (SU6656), and Ariad Pharmaceuticals (AP23464 and AP 23451) each have c-Src inhibitors in pre-clinical or Phase I clinical trials—the efficacy, mechanism of action, and clinical utility of these compounds in mediating molecular effects downstream of c-Src remain to be seen.
A few tyrosine phosphorylation sites on signaling proteins downstream of c-Src have been reported, including the non-receptor tyrosine kinase FAK, the adaptor proteins p130 CAS and Sam68, the actin binding protein cortactin, the phospholipid binding protein annexin A2 and the STAM interacting protein Hrs. See Calalb et al., Mol. Cell. Biol. 15: 954-963 (1995); Belsches et al., Front. Biosci. 2: d501-518 (1997); Bache et al., Eur. J. Biochem 269: 3881-3887 (1997); Schaller et al., Mol. Cell. Biol. 14: 1680-1688 (1994); Shen et al., Oncogene 18: 4647-4653 (1999). Nonetheless, the small number of c-Src signaling pathway-related phosphorylation sites that have been identified to date do not facilitate a complete and accurate understanding of how protein activation downstream of c-Src is driving the progression of cancers in which this kinase is activated.
Accordingly, there is a continuing need to unravel the molecular mechanisms of c-Src driven oncogenesis by identifying the downstream signaling proteins mediating cellular transformation in diseases involving activated c-Src. Identifying particular phosphorylation sites on such signaling proteins and providing new reagents, such as phospho-specific antibodies and AQUA peptides, to detect and quantify them remains particularly important to advancing our understanding of the biology of these cancers.
Presently, a handful of compounds targeting c-Src are in or entering clinical trials for the treatment of cancer. Although the activation and/or expression of c-Src itself can be detected, it is clear that other downstream effectors of c-Src signaling, having diagnostic, predictive, or therapeutic value, remain to be elucidated. Accordingly, identification of downstream signaling molecules and phospho-sites involved in the progression of c-Src driven cancers, and development of new reagents to detect and quantify these sites and proteins, may lead to improved diagnostic/prognostic markers, as well as novel drug targets, for the detection and treatment of these diseases.